CN112359039A - shRNA sequence expressed by targeted silencing BRD4 gene and application thereof - Google Patents
shRNA sequence expressed by targeted silencing BRD4 gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, and particularly relates to a shRNA sequence expressed by a targeted silent BRD4 gene and application thereof. The invention designs and synthesizes shRNA molecules based on the mRNA sequence of the BRD4 gene, constructs the oligonucleotide sequence of shRNA, and the shRNA sequence infects human small cell lung cancer cells after being packaged into lentivirus by a lentivirus vector, can be combined with the mRNA of the BRD4 gene, efficiently interferes with the transcription and translation of the gene, reduces the expression of the BRD4 gene in the small cell lung cancer cells, and inhibits the proliferation of the small cell lung cancer cells. The shRNA sequence provided by the invention provides a theoretical basis for a new strategy for treating small cell lung cancer by taking a BRD4 inhibitor as a core, is expected to be applied to the preparation of an anti-cancer gene drug with high efficiency, strong specificity and small side effect, especially the research and development of small cell lung cancer gene drugs, and has great social and economic benefits.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a shRNA sequence expressed by a targeted silent BRD4 gene and application thereof.
Background
Lung cancer is the most common malignant tumor at present, and the death rate of lung cancer is the first of malignant tumors. Lung cancer can be divided into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC), the latter accounting for approximately 12-14% of all lung cancer cases. One of the most significant features of SCLC is the high mutation frequency of some important genes, such as the mutation rate of tumor protein 53 gene (TP 53) is 75% -90%, and the mutation rate of retinoblastoma 1 (RB 1) gene is close to 100%. Currently, the most common treatment methods for SCLC clinically include surgery, chemotherapy, and radiotherapy. SCLC is sensitive to chemotherapy and radiotherapy in the initial stage, but because SCLC doubling time is short, malignancy is high, drug resistance and relapse are very easy to occur, and most patients have short survival period after diagnosis and poor prognosis. At present, compared with other tumors, the treatment mode of SCLC is still relatively limited and has no obvious effect on improving the survival time of patients. Therefore, there is an urgent need to find more safe and effective drug targets to improve the survival of SCLC patients.
Bromodomain protein 4 (BRD 4) is located in chromosome 19p13 region and is an important functional protein of the bromodomain and superterminal domain families. In SCLC, BRD4 can bind directly to the enhancer of the glabrous lepidoptera complex-like 1 (ASCL 1) gene, thereby promoting the development of SCLC; the expression level of ASCL1 correlates with the sensitivity of SCLC cells to the inhibitor JQ1 of BRD 4. The BRD4 inhibitor ABBV-075 can induce Caspase3/7 activity, promote the expression of pro-apoptotic protein BIM, promote the formation of Bcl2-BIM complex and the like to trigger apoptosis.
RNA interference technology (RNAi) is a technology that relies on short sequences specific for double-stranded RNA to achieve post-transcriptional gene silencing. Currently, RNAi technologies commonly used in laboratories mainly include siRNA oligonucleotide vectors and shRNA lentiviral plasmid expression vectors. Short hairpin ribonucleic acid (shRNA) can be stabilized by virus-mediated transfection, and can reduce off-target effects.
The shRNA is firstly inserted into a lentiviral vector to form a recombinant lentiviral plasmid, the lentiviral plasmid and other helper plasmids form lentivirus by means of 293FT cells, and finally the cells are transfected by the lentivirus to play the silencing effect of the shRNA. Under the action of an endonuclease Dicer, shRNA is split into 21-25nt nucleotide chains consisting of a sense chain and an antisense chain. The antisense strand is combined with a specific enzyme to form a silencing complex RISC (the RISC complex contains siRNA, exonuclease, endonuclease, helicase and other elements), which is induced by RNA, the nucleotide double strand is depolymerized into two single strands by the activated RISC, then the antisense strand recognizes and is combined with target mRNA which is homologous with the nucleotide double strand, the activated RISC cuts the specific position of the target mRNA under the guide of the antisense strand, and simultaneously the cut mRNA is specifically degraded by the enzyme in the RISC complex, thereby blocking the genetic information transmission of the mRNA. However, how to use the shRNA technology to research shRNA specifically aiming at small cell lung cancer cells has important significance for preventing or treating small cell lung cancer.
Disclosure of Invention
In view of the above, the present invention aims to provide shRNA sequences for targeted silencing of BRD4 gene and applications thereof. According to the influence of the silenced BRD4 gene on the proliferation of the small cell lung cancer, the specificity of the shRNA sequence of the silenced BRD4 gene is judged, and the inhibition effect of the shRNA sequence of the silenced BRD4 gene on the small cell lung cancer cell is verified.
In order to realize the purpose, the invention adopts the technical scheme that:
the invention provides an shRNA sequence expressed by a targeted silent BRD4 gene, wherein the shRNA is marked as shBRD4-1 and shBRD4-2, the sequence of the shBRD4-1 is shown as SEQ ID NO.1, and the sequence of the shBRD4-2 is shown as SEQ ID NO. 2.
The invention also provides an oligonucleotide sequence for preparing the shBRD4-1, wherein a sense chain shBRD4-1-F is shown as SEQ ID NO.3, namely: 5 '-CCGGTgccaacgcagccagcaccaacCTCGAGgttggtgctggctgcgttggcATTTTT-3', and the antisense strand shBRD4-1-R is as shown in SEQ ID No.4, namely: 5 '-AAAAATgccaacgcagccagcaccaacCTCGAGgttggtgctggctgcgttggcACCGG-3';
the invention also provides an oligonucleotide sequence for preparing the shBRD4-2, wherein a sense chain shBRD4-2-F is shown as SEQ ID NO.5, namely: shBRD 4-2-F: 5 '-CCGGTagcagctcaagtgctgcagcgCTCGAGcgctgcagcacttgagctgctATTTTT-3', and the antisense strand shBRD4-2-R is as shown in SEQ ID No.6, namely: 5 '-AAAAATagcagctcaagtgctgcagcgCTCGAGcgctgcagcacttgagctgctACCGG-3'.
In some embodiments, the invention also provides a lentiviral expression vector comprising an shRNA sequence for targeted silencing of BRD4 gene expression as described above, wherein the vector is pLKO.1-TRC-shBRD4-1 or pLKO.1-TRC-shBRD 4-2. The lentiviral expression vector is used for expressing the shRNA sequence of claim 1, and comprises a target site sequence shBRD4-1 or shBRD4-2 for silencing BRD4 of a target gene.
Further, the lentiviral expression vector adopts primer annealing to synthesize shBRD4-1 or shBRD4-2 sequences; the method comprises the steps of cloning a synthesized shBRD4-1 or shBRD4-2 sequence into a lentivirus expression vector pLKO.1-TRC to obtain the shBRD4-1 primer sequence shown as SEQ ID No. 3-4, and the shBRD4-2 primer sequence shown as SEQ ID No. 5-6.
In some embodiments, the invention also provides application of the shRNA sequence for silencing BRD4 gene expression in preparation of a medicine for inhibiting BRD4 gene expression, wherein the application is in preparation of a medicine for treating/preventing small cell lung cancer.
The invention also provides a medicine for treating small cell lung cancer, which comprises the shRNA sequence expressed by the targeted silent BRD4 gene.
Compared with the prior art, the invention has the beneficial effects that:
the invention designs and synthesizes shRNA molecules based on the mRNA sequence of the BRD4 gene, constructs the oligonucleotide sequence of shRNA, and the two pairs of shRNA sequences infect human small cell lung cancer cells after being packaged into lentivirus by a lentivirus vector, can be combined with the mRNA of the BRD4 gene, efficiently interfere the transcription and translation of the gene, reduce the expression of the BRD4 gene in the small cell lung cancer cells, and inhibit the proliferation of the small cell lung cancer cells. The shRNA sequence designed by the invention limits and inhibits the growth rate of small lung cancer cell H446, and has very important significance for treating small lung cancer. Provides theoretical basis for a new strategy for treating the small cell lung cancer by taking a BRD4 inhibitor as a core, is expected to be applied to the preparation of an anti-cancer gene medicine with high efficiency, strong specificity and small side effect, especially aims at the research and development of the small cell lung cancer gene medicine, and has great social and economic benefits.
Drawings
FIG. 1 shows that Western blot detects the silencing of BRD4 expression by shBRD 4;
fig. 2 is a graph showing the effect of shBRD4 on the proliferation of human small cell lung cancer cell H446.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative of the present invention and are not intended to limit the scope of the invention. One skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
In the following examples, the materials and reagent portions used were derived as follows:
lentiviral packaging vectors pLP1, pLP2, pLPSVG, and lentivirus knockdown plasmid vector Plko.1-TRC were all purchased from Addgene, USA. Human small cell lung cancer cells H446 used in the present invention are purchased from American Type Culture Collection (ATCC).
The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. The invention adopts the prior art in the field except for special notes.
Example 1: construction of Lentiviral vectors
The mRNA sequence of the human BRD4 gene (NM-058243) is obtained according to a GenBank database (http:// www.ncbi.nlm.nih.gov/GenBank), the shRNA aiming at the human BRD4 gene is designed according to the shRNA design principle, and target sequences of the shRNA are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 and are used for silencing the human BRD4 gene. SEQ ID No.1 is: 5 '-gccaacgcagccagcaccaac-3', the corresponding coding region being NM-001330384.1 (nucleotide position 400-420); SEQ ID No.2 is: 5 '-agcagctcaagtgctgcagcg-3', the corresponding coding region being XM _011527854.2 (nucleotide position 1126) -1146). Two pairs of complementary oligonucleotide sequences shBRD4-1-1 and shBRD4-1-2 are designed and synthesized according to the two target sequences. Wherein, the sense chain shBRD4-1-F of the shBRD4-1-1 is shown as SEQ ID NO.3, namely: 5 '-CCGGTgccaacgcagccagcaccaacCTCGAGgttggtgctggctgcgttggcATTTTT-3'; the antisense chain shBRD4-1-R is shown as SEQ ID NO.4, namely: 5 '-AAAAATgccaacgcagccagcaccaacCTCGAGgttggtgctggctgcgttggcACCGG-3';
the sense strand shBRD4-2-F of shBRD4-1-2 is shown as SEQ ID NO.5, namely: 5 '-CCGGTagcagctcaagtgctgcagcgCTCGAGcgctgcagcacttgagctgctATTTTT-3', and the antisense strand shBRD4-2-R is as shown in SEQ ID No.6, namely: 5 '-AAAAATagcagctcaagtgctgcagcgCTCGAGcgctgcagcacttgagctgctACCGG-3'. The sequences described in the present invention are all synthesized by Biotechnology engineering (Shanghai) GmbH.
The 4 double-stranded oligonucleotide short fragments were ligated to the double-digested lentiviral vector Plko.1-TRC, respectively. Transforming the ligation product into a DH5 alpha escherichia coli competent cell by a heat shock method, culturing for 16h at 37 ℃ on an LB solid culture medium containing benzyl resistance, selecting a single colony, shaking the single colony in an LB liquid culture medium containing benzyl resistance at 37 ℃, rotating at 220rpm for 16h, collecting thalli to extract plasmids, carrying out enzyme digestion to verify the plasmids, and sending the enzyme-digested positive plasmids to Huada gene sequencing for verification. Sequencing showed that the successfully constructed lentiviral plasmid was used in subsequent experiments.
Through the method, lentiviral vectors pLKO.1-TRC-shBRD4-1 and pLKO.1-TRC-shBRD4-2 containing the target plasmids are respectively constructed.
Example 2: preparation of cell lentivirus and lentivirus transfected cell
Lentiviruses were packaged with reference to Lipofectamine 2000 reagent instructions. Two kinds of lentivirus vector transfected cells prepared in example 1 were used as experimental groups, and pLKO.1-TRC lentivirus empty vector transfected cells were used as control groups. Lipofectmin2000 the objective plasmid and helper plasmids pLP1, pLP2 and pLPSVG were transferred into 293FT cells, virus was prepared and collected, then human small cell lung cancer cells H446 were infected with lentivirus containing the target shBRD4 sequence, and puromycin (puromycin) was used to select resistant cells for experiments.
0.75. mu.g of pLP1, 0.35. mu.g of pLP2, 0.49. mu.g of pLPSVG, and 0.61. mu.g of lentiviral vector pLKO.1-TRC-shBRD4-1 were added to 0.5mL of low serum OPTI-MEM medium, and gently mixed, and incubated at room temperature for 5min to obtain solution A for use.
Adding 9 μ L liposome Lipofectmin2000 into 0.5mL OPTI-MEM medium, mixing, and standing at room temperature for 5min to obtain solution B.
Mixing solution A and solution B at a ratio of 1:1 to obtain mixed solution, and incubating at room temperature for 20 min; 293FT cells were digested and counted under a microscope with cell density adjusted to one third of the area of the bottom of the dish. The 293FT cell suspension and the mixed solution are mixed gently and fully according to the ratio of 1:1, transferred into a culture dish and shaken gently to mix evenly and pave the bottom. After the culture dish is placed in an incubator to be incubated for 12 hours, the cells are changed in liquid, and the culture is continued. After two days of culture, the cell supernatant was aspirated by a 5mL sterile syringe, and the cell supernatant impurities were filtered off with a 0.45 μm microporous membrane to collect the virus. The method for packaging lentivirus by using lentivirus vector pLKO.1-TRC-shBRD4-2 and lentivirus empty vector pLKO.1-TRC is identical with the method for packaging lentivirus by using lentivirus vector pLKO.1-TRC-shBRD 4-1.
H446 cells were transfected separately with the harvested virus: on the first day, plating a plate, counting cells, and adjusting the cell density to be one third of the bottom area of a culture dish of 6 cm; the next day, virus infection, 6cm petri dish removed, supernatant discarded, virus and H446 cell culture medium 1:1, mixing and adding 8mg/mL polybrene (polybrene) to enable the action concentration to be 8ug/mL, putting the mixture into an incubator, and incubating for 8 hours; after incubation for 8h, removing the supernatant, replacing the cell with a liquid, and continuously putting the cell into an incubator for two days; after two days of incubation, the supernatant was discarded and the cells were incubated with 1000mg/mL puromycin at 1: diluting 1000, and screening for 2-3 days; reseeding: the cells after two days of screening are digested and reseeded back to the culture dish to obtain the resistant living cells for subsequent experiments.
Example 3: western blot detection of protein expression level of BRD4 gene in H446 cells
Collecting virus infected H446 cells, extracting total protein, determining protein concentration, running gel after protein denaturation to transfer a membrane, sealing the membrane for 1H by 5% skimmed milk, incubating overnight by using BRD4 antibody and beta-actin antibody, incubating for 1H at room temperature by using goat anti-mouse antibody connected with horseradish peroxidase, washing the membrane for 3 times at room temperature by using TBS-T solution after antibody incubation, each time for 4min, finally placing the membrane in a dark box, taking the dark box into a dark room, exposing, fixing the membrane during exposure, and blotting a protein strip on the membrane under the action of luminous liquid and developing liquid. Collecting the H446 cells surviving the experimental group and the control group after puromycin screening, respectively extracting the total cell protein, and detecting the expression condition of the target protein BRD4 in different groups of cells by Western blotting. FIG. 1 is a Western blotting graph showing the expression level of shBRD4 on BRD4 gene; in the figure, beta-actin is an internal reference protein, Vector is a control group infected with pLKO.1-TRC empty Vector, and shBRD4-1 and shBRD4-2 are experimental groups infected with Plko.1-TRC-shBRD4-1 and Plko.1-TRC-shBRD4-2, respectively. As can be seen from FIG. 1, compared with the Vector in the control group, both shBRD4-1 and shBRD4-2 can significantly silence the expression of BRD4 in H446 cells, and have significant silencing effect on the expression of BRD4 in H446 cells.
Example 4: MTT experiment for detecting influence of shBRD4 on H446 cell proliferation
(1) And (3) treating the cells: collecting virus-infected H446 cells, digesting with pancreatin, centrifuging at 200 g for 5min, resuspending the medium, counting the cells using a cell counting plate, using a 96-well plate, containing 4X 10 of medium per 100. mu.L of the well according to the cell growth characteristics3One cell, three parallel wells.
(2) MTT detection: after the plate is planted, 10 mu L of MTT solution of 5 mg/mL is added into a hole to be detected every 24H, the plate is incubated for 1.5H in an incubator, blue-purple formazan crystals are observed under a microscope, H446 cells directly absorb and discard supernatant, 100 mu L of DMSO is added into each hole to dissolve the crystals, the solution is evenly tapped gently, the absorbance value of each hole is measured at the wavelength of 550 nm by using an enzyme labeling instrument, and the relative survival rate of the cells is calculated.
FIG. 2 is a graph showing the effect of shBRD4 on H446 cell proliferation, with the vector group being the control group and the shBRD4-1 and shBRD4-2 groups being the experimental group. As shown in figure 2, the shBRD4-1 and shBRD4-2 groups can both remarkably inhibit the proliferation of small cell lung cancer cells H446, so that the shRNA designed by the invention can be used for preparing a medicine for treating small cell lung cancer.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> university of Jiangsu
<120> shRNA sequence for targeted silencing BRD4 gene expression and application thereof
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<213> human source (Homo sapiens)
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agcagctcaa gtgctgcagc g 21
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<213> human source (Homo sapiens)
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<213> human source (Homo sapiens)
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<213> human source (Homo sapiens)
<400> 5
ccggtagcag ctcaagtgct gcagcgctcg agcgctgcag cacttgagct gctattttt 59
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<213> human source (Homo sapiens)
<400> 6
aaaaatagca gctcaagtgc tgcagcgctc gagcgctgca gcacttgagc tgctaccgg 59
Claims (8)
1. An shRNA sequence expressed by a targeted silent BRD4 gene is characterized by being marked as shBRD4-1 and shBRD 4-2; the sequence of the target site of the shBRD4-1 is shown as SEQ ID NO.1, and the sequence of the target site of the shBRD4-2 is shown as SEQ ID NO. 2.
2. An shRNA sequence according to claim 1, wherein the shBRD4-1 oligonucleotide sequence is prepared such that the sense strand sequence is shown as SEQ ID No.3 and the antisense strand sequence is shown as SEQ ID No. 4.
3. An shRNA sequence according to claim 1, wherein the shBRD4-2 oligonucleotide sequence is prepared such that the sense strand sequence is shown as SEQ ID No.5 and the antisense strand sequence is shown as SEQ ID No. 6.
4. A lentiviral expression vector for expressing the shRNA sequence of claim 1, wherein the lentiviral expression vector comprises a target site sequence shBRD4-1 or shBRD4-2 for silencing BRD4 of a target gene.
5. The method for constructing a lentiviral expression vector according to claim 4, wherein the lentiviral expression vector is annealed by a primer to synthesize a shBRD4-1 or shBRD4-2 sequence; the method comprises the steps of cloning a synthesized shBRD4-1 or shBRD4-2 sequence into a lentivirus expression vector pLKO.1-TRC to obtain the shBRD4-1 primer sequence shown as SEQ ID No. 3-4, and the shBRD4-2 primer sequence shown as SEQ ID No. 5-6.
6. The application of the shRNA sequence according to claim 1 in preparing a medicament for inhibiting the expression of the BRD4 gene.
7. The application of the shRNA sequence according to claim 1 in preparing medicines related to treating/preventing small cell lung cancer.
8. A medicament for treating small cell lung cancer, comprising the shRNA sequence targeted to silence BRD4 gene expression of claim 1.
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| CN114045264A (en) * | 2021-11-16 | 2022-02-15 | 珠海中科先进技术研究院有限公司 | Method for obtaining driving element |
| CN114107293A (en) * | 2021-10-26 | 2022-03-01 | 江苏大学 | shRNA for specifically inhibiting KSR1 gene expression and application thereof |
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| Title |
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| RYAN LENHART 等: "Sensitivity of Small Cell Lung Cancer to BET Inhibition Is Mediated by Regulation of ASCL1 Gene Expression", 《SMALL MOLECULE THERAPEUTICS》 * |
| YUN-FEI LIAO 等: "High level of BRD4 promotes non-small cell lung cancer progression", 《ONCOTARGET》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107293A (en) * | 2021-10-26 | 2022-03-01 | 江苏大学 | shRNA for specifically inhibiting KSR1 gene expression and application thereof |
| CN114045264A (en) * | 2021-11-16 | 2022-02-15 | 珠海中科先进技术研究院有限公司 | Method for obtaining driving element |
| CN114045264B (en) * | 2021-11-16 | 2024-05-14 | 珠海中科先进技术研究院有限公司 | Method for acquiring driving element |
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